Stiff bundles of actin filaments provide structural support for fingers of membrane such as microvilli, stereocilia, and filopodia. One model system for studying the structure, function, and assembly of actin bundles is the crystalline bundle in the acrosomal process of Limulus sperm. In unactivated sperm, the bundle is coiled around the base of the nucleus but when the sperm contacts the egg, the actin bundle straightens and extends to its full 80 mum length in a few minutes. The acrosomal process is a simple structure, composed of actin and the actin crosslinking protein scruin, a 102 kDa polypeptide complexed with two 17 kDa polypeptides. The 13 A resolution 3D structure of a filament in the acrosomal process scruin is organized into two domains which bind to two actin subunits. The sequence of the 102 kDa polypeptide is divided into a tandem pair of homologous domains each containing a six-fold repeat of a 50 residue sequence. A search of the structure database reveals this sequence corresponds to a four-stranded beta-sheep motif which are repeated five, six, or seven times for form a disc-shaped beta-flower domain. This domain is found in viruses, procaryotes, and eucaryotes. In addition to scruin, the beta-flower domain is also found in Drosophila ovary kelch, C. elegans sperm spe26, and mouse placental MIPP. Because these proteins have not been isolated, their functions have not been studied but their similarity with scruin suggests a cytoskeletal function. Our goal is to describe how scruin crosslinks actin filaments into a bundle. To accomplish this goal we have two specific aims. The first aim is to describe completely the structure and function of scruin. Biochemical and molecular biology experiments will describe the organization of scruin domains, their association with the 17 kDa polypeptide, the binding sites on actin, and the actin binding sites on scruin. The second goal is to determine the 3D structure of the actin bundle in collaboration with the cryoEM group at Baylor. We will provide them with actin filaments decorated with scruin domains, specific anti- scruin antibodies, and scruin labelled at specific residues with gold clusters. Using the technique of image reconstruction from cryoelectron micrographs, the Baylor group will map the location of residues in the sequence to the 3D structure. This information will be crucial to later studies to determine the structure of the filament to a resolution of 6A. Because scruin and its related proteins are involved in some aspect of gametogenesis, our studies may be relevant to studies in human reproduction and early development.